Oxidized thymosin β4

ABSTRACT

The present invention relates to the use of oxidized thymosin β4 in therapy, more particularly in the treatment of diseases or conditions associated with an inflammatory response of septic shock.

The present invention relates to a peptide factor isolated fromsteroid-treated monocytes. More particularly the invention relates to apeptide factor which can be used to replace steroid therapy.

Steroids are effectively used for anti-inflammatory diseases, such asasthma, eczema, allergic reactions, and rheumatic diseases such asrheumatoid arthritis. However, steroids have serious side effects andare therefore only used in cases where non-steroidal anti-inflammatorydrugs are not effective.

Monocytes are important immune effector cells that play a fundamentalrole in cellular immunity. In addition to their antigen-presenting andphagocytic activities at the sites of inflammation, peripheral bloodmononuclear cells are also involved in the synthesis and release of avariety of pro-inflammatory enzymes and polypeptide cytokines whichmodulate neutrophil responses. The production of these components can besuppressed by glucocorticoids and this has been suggested as the basisfor their anti-inflammatory action.

The effect of steroid-induced factors on neutrophil migration isprimarily of interest in elucidating anti-inflammatory mechanisms.Corticosteroids down regulate the synthesis of many pro-inflammatorymediators (Lew et al 1988; Almawi et al 1991; Standford et al 1992) butsome of their actions can be interpreted in terms of up-regulation ofanti-inflammatory mediators.

The neutrophil migration stimulating activity of steroid induced factorssuggests that dispersive locomotion tends to prevent cells collecting ata focus and this may be important in terminating inflammatory responses.

Stevenson (1973, 1974, 1978) demonstrated that human monocytes whenincubated in the presence of antiinflammatory corticosteroids released aprotease sensitive factor that enhanced the migration of neutrophilsfrom a cell pellet contained in a short capillary tube.

Later studies demonstrated that the phenomenon of stimulated neutrophilmigration was also observed with leucocytes from patients receivingsteroid therapy.

Recently, Chettibi et al (1993, 1994) have investigated the steroidinduced stimulatory effect on neutrophil migration using an automatedcell tracking assay enabling study of the behaviour of cells migratingon protein-coated glass coverslip.

These Studies Determined:

1. Steroid-treated monocyte supernatant (STMS) causes a dramaticincrease in the speed of locomotion of human neutrophils and asignificant decrease in their adhesion to protein-coated glass. Incontrast, control monocyte supernatants have a smaller effect on thespeed of locomotion, but cause a large increase in adhesiveness.

2. The supernatant activity was produced equally well in the presence orabsence of serum after 24 h culture at 37° C. with 10⁻⁶M dexamethasone.

3. The effect of the steroid-treated monocyte supernatant on the speedof locomotion of human peripheral blood neutrophils was not altered byrabbit polyclonal antisera against lipocortins 1-6.

4. Rabbit anti-interleukin-8 antibody which blocked the effect of IL-8on the speed of locomotion of neutrophils did not antagonize thelocomotion stimulating action of steroid-treated monocyte supernatant.

5. The exocellular release of this factor(s) by human mononuclearleucocytes suggests that it may be an in vivo mediator of theanti-inflammatory effect of glucocorticoids.

However, there is no disclosure of what the active agent(s) in STMSmight be.

Huff T. et al. (1995) and Heintz D. et al. (1994) describe studiesinvolving beta-thymosins and how they interact with G-actin in abiomolecular complex and inhibit the polymerisation to F-actin underhigh salt conditions. The oxidised form of thymosin β4 is disclosed asinhibiting actin polymerisation, however, only at a 20-fold higherconcentration than thymosin β4. Neither document however implicates anymedical role for oxidised thymosin β4. In fact the papers appear toteach away from a positive role for oxidised thymosin β4.

U.S. Pat. No. 5,578,570 (Goldstein et al.) discloses a method oftreating septic shock by administering thymosin β4. There is nodisclosure however of oxidised thymosin β4 or suggestion that this mayhave a role in treating septic shock.

It is an object of the present invention to provide a replacement tosteroid therapy.

The present invention is based in part on the observations by thepresent inventors that the factor associated with neutrophil locomotionis an oxidised form goof thymosin β4.

According to a first aspect the present invention provides use ofoxidised thymosin β4 or physiologically active variant thereof intherapy.

Typically oxidised thymosin β4 is a form of thymosin β4 in which amethionine residue, 6 amino acids from the N-terminus, (Met6), isoxidised such that the residue is converted to methionine sulphoxide.Moreover, the methionine residue (Met6) may be further oxidised to themethionine sulphone and this as such is also encompassed by the presentinvention. Other modifications of the methionine residue may also beevisaged, such as complexing the sulphur with metals, which may resultin an active form of thymosin β4 similar to the oxidised form describedherein.

It is understood that the oxidised thymosin β4 may be obtained forexample by reacting native thymosin β4 under oxidising conditions, forexample by treating with hydrogen peroxide, to form oxidised thymosinβ4. Thus native thymosin β4 may first be obtained and thereafteroxidised to the oxidised form.

It has been observed that samples of native thymosin β4 may contain lowlevels, such as 10%, of oxidised thymosin β4 thought to be as a resultof auto-oxidation. The present inventors however are the first toassociate the oxidised form of thymosin β4 with a physiologicalactivity. Generally speaking therefore the present invention providesthe use of purified oxidised thymosin β4. Typically the presentinvention provides use of preparations of purified oxidised thymosin β4which comprise at least 30%, preferably 60%, more preferably 80%, mostpreferably 90%, oxidised thymosin β4 with the residual portionaccounting for non-oxidised thymosin β4. Preferably however thepreparations of oxidised thymosin β4 comprise substantially all oxidisedthymosin β4 (ie. substantially no non-oxidised thymosin β4).

Thymosin β4 in an oxidised or non-oxidised form may be obtained from anysuitable source, for example from steroid treated monocytes. Moreover,the thymosin β4 may be derived from any suitable species, but istypically of mammalian origin, such as bovine, equine, murine or humanorigin. It is to be noted that bovine, equine, murine, rat and humanthymosin β4 are all identical in sequence. Thus, for example, bovinethymosin β4 may provide a suitable source of thymosin β4 for subsequentoxidation and administration to other species, such as humans.

It is understood that physiologically active variants of the oxidisedthymosin β4 are variants which display the same or similar physiologicalproperties as the oxidised thymosin β4. It is to be preferred that suchvariants would include the oxidised methionine, but may be truncated,deleted or mutated forms thereof.

It will be understood that for the particular oxidised thymosin β4embraced herein, variations (natural or otherwise) can exist. Thesevariations may be demonstrated by (an) amino acid difference(s) in theoverall sequence or by deletions, substitutions, insertions, inversionsor additions of (an) amino acid(s) in said sequence. All suchderivatives are included within the scope of this invention providedthat the derivatives are physiologically active (ie. display oxidisedthymosin β4 activity as defined herein). For example, for the purpose ofthe present invention conservative replacements may be made betweenamino acids, within the following groups:

(I) alanine, serine and threonine;

(II) glutamic acid and aspartic acid;

(III) arginine and lysine;

(IV) asparagine and glutamine;

(V) isoleucine, leucine and valine;

(VI) phenylalanine, tyrosine and tryptophan.

(VII) methionine and other methionine analogues

(VIII) methionine and other methionine analogues where the sulphur isreplaced by Group VIB elements (e.g. Selenium, Tellurium, Polonium).

(IX) oxidised methionine and other oxidised methionine analogues (e.g.Group VIB analogues, methionine sulphoximine).

(X) methionine and other sulphur-containing amino acids (e.g. cysteine)including their oxidised analgoues.

Early molecular modelling studies suggest that the methionine residue(met-6) is at the top of one of three helices in the peptide. Molecularmodelling should help identify a shorter peptide which may have theactivity observed for STMS and oxidised thymosin β4 and would be apreferred molecule to use in preparing pharmaceuticals withanti-inflammatory activity.

Indeed this may assist in the development of peptide mimetics whichdisplay the same physiological function as the oxidised thymosin β4.

Moreover, it may be possible to increase the half life of oxidisedthymosin β4 or physiologically active variants thereof by use ofappropriate chemical modification (eg. acetylation) or use of D aminoacids.

The isolated oxidised thymosin β4 may have a blocked N-terminal.

According to the present invention there is also provided a syntheticoxidised thymosin β4 comprising the peptide sequence of thymosin β4 inoxidised form or physiologically active variant thereof.

The synthetic oxidised thymosin β4 may be modified and/or amino acidsubstituted as described above, as long as the physiological activityremains. For example seleno-methionine could be introduced in place ofmethionine and oxidised in the same manner.

The invention further provides the use of an oxidised peptide asdescribed herein in the preparation of a medicament for the treatment ofa chronic or acute inflammatory condition. Such inflammatory conditionsinclude Inflammatory Arthropathies such as Rheumatoid arthritis,Psoriatic arthritis, Crystal arthritis, Reactive arthritis, Ankylosingspondylitis, Infectious arthritis, Juvenile chronic arthritis;Connective Tissue Diseases, such as Systemic Lupus Erythematosis,Sjogren's Syndrome, Polymyalgia Rheumatica, Cranial arteritis;Vasculitic Syndromes, such as Wegener's Granulomatosis, PolyarteritisNodosa, Churg Strauss Syndrome; Respiratory Diseases, such as Asthma,Chronic Obstructive Pulmonary Disease, Fibrosing Alveolitis,Hypersensitivity Pneumonitis, Sarcoidosis, Allergic aspergillosis,Cryptogenic pulmonary eosinophilia, Bronchiolitis obliterans organisingpneumonia; Dermatological Diseases, such as Inflammatory dermatosisincluding psoriasis, Eczema, Urticaria; Gastro-intestinal Diseases, suchas Ulcerative Colitis, Crohn's Disease, Lupoid hepatitis; HaematologicalDisease, such as Haemolytic anaemia, Idiopathic ThrombocytopenicPurpura, Multiple Myeloma, Lymphoma/leukaemia;Transplantation/Prosthetics, such as Graft rejection, Graft versus hostdisease, Tissue reaction to implanted prostheses; and Infections, suchas Tuberculosis, Malaria Pneumocystis carinii pneumonia, Leprosy.

Moreover, oxidised thymosin β4 may be administered in conjunction withother drugs, eg. cytokines such as interferon which may induce aninflammatory response as a side effect. Thus, in one aspect oxidisedthymosin β4 may serve to minimise or reduce physiological or diseasestates which are characterised in part by inappropriate inflammation.

Additionally, it should be appreciated that the uses of oxidisedthymosin β4 mentioned above do not only extend to human conditions.Thus, oxidised thymosin β4 may be used in the treatment of animals suchas cats, dogs, horses, cows, sheep, pigs and goats with similarconditions to those mentioned above.

The present invention further provides the use of oxidised thymosin β4in the preparation of a medicament for the treatment of septic shock.Typically the oxidised thymosin β4 is in a purified form as describedabove.

The invention further provides a pharmaceutical composition comprisingoxidised thymosin β4 as described herein.

The invention further provides use of a nucleotide molecule having asequence capable of encoding thymosin β4 as described herein forsubsequently preparing oxidised thymosin β4.

In a particular embodiment the invention provides the use of a vector orvectors comprising the nucleotide molecule in the preparation ofoxidised thymosin β4 and trancated, deleted and mutated forms thereof asdescribed herein.

Alternatively the present invention provides the use of a vector orvectors comprising the nucleotide molecule in the preparation of amedicament comprising oxidised thymosin β4 and trancated, deleted andmutated forms thereof for the treatment of a inflammatory condition.

The use of oxidised thymosin β4 as described herein in place of steroidtreatment will alleviate the side effects which are normally associatedwith the use of steroids.

The oxidised thymosin β4 can be used for treatment of patients where nonsteroidal anti inflammatory drugs are currently used as an alternativeto steroids because of the risks of side-effects.

Use of highly purified oxidised thymosin β4 or of synthetic or expressedthymosin β4 which is subsequently oxidised will be safe and reliable,since it will generally not be foreign to the body to which it is beingadministered.

Accurate amounts can be administered.

The amount of oxidised thymosin β4 required to be effective in atreatment will, of course, vary and is ultimately at the discretion ofthe medical or veterinary practitioner. The factors to be consideredinclude the condition being treated, the route of administration, andnature of the formulation, the recipients body weight, surface area, ageand general condition, and the particular compound to be administered. Asuitable effective dose may lie in the range of about 0.001 to about 120mg/kg bodyweight, e.g. 0.01 to about 120 mg/kg body weight, preferablyin the range of about 0.01 to 50 mg/kg, for example 0.05 to 20 mg/kg.The total daily dose may be given as a single dose, multiple doses,e.g., two to six times per day or by intravenous infusion for selectedduration. For example, for a 75 kg mammal (e.g. a human) the dose rangemay be about 8 to 9000 mg per day, and a typical dose could be about 50mg per day. If discrete multiple doses are indicated treatment mighttypically be 15 mg of oxidised thymosin β4 given up to 4 times per day.

Whilst it is possible for the active compound to be administered alone,it is preferable to present the active compound in a pharmaceuticalformulation. Formulations of the present invention, for medical use,comprise oxidised thymosin β4, or a salt thereof together with one ormore pharmaceutically acceptable carriers and optionally othertherapeutic ingredients. The carrier(s) should be pharmaceuticallyacceptable in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient.

The present invention, therefore, further provides a pharmaceuticalformulation comprising oxidised thymosin β4 or a pharmaceuticallyacceptable salt or physiologically functional derivative thereoftogether with a pharmaceutically acceptable carrier therefor.

There is also provided a method for the preparation of a pharmaceuticalformulation comprising bringing into association oxidised thymosin β4 ora pharmaceutically acceptable salt or physiologically functionalderivative thereof, and a pharmaceutically acceptable carrier therefor.

Formulations according to the present invention include those suitablefor oral, nasal, topical, vaginal, rectal or parenteral (includingsubcutaneous, intraarthrodial (ie. within joints) intramuscular andintravenous) administration including biolistic eg. Powderject®administration. Preferred formulations are those suitable for oral,topical or parenteral administration.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier or a finely divided solid carrier or both and then, ifnecessary, shaping the product into desired formulations.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units as capsules, cachets, tablets,lozenges, comprising the active ingredient in a flavoured base, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert base such as gelatin and glycerin, or sucrose andacacia; and mouth-washes comprising the active ingredient in a suitableliquid carrier. Each formulation generally contains a predeterminedamount of the active compound; as a powder or granules; or a solution orsuspension in an aqueous or non-aqueous liquid such as a syrup, anelixir, an emulsion or draught and the like.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed, tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform such as a powder or granules, optionally mixed with a binder, (e.g.povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inertdiluent, preservative, disintegrant (e.g. sodium starch glycollate,cross-linked povidone, cross-linked sodium carboxymethyl cellulose),surface active or dispersing agent. Moulded tablets may be made bymoulding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethylcellulose in varying proportions to provide thedesired release profile.

A syrup may be made adding the active compound to a concentrated,aqueous solution of a sugar, for example sucrose, to which may also beadded any accessory ingredients. Such accessory ingredient(s) mayinclude flavourings, an agent to retard crystallization of the sugar oran agent to increase the solubility of any other ingredients, such as apolyhydric alcohol for example glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier such as cocoa butter.

Formulations suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the active compound which ispreferably isotonic with the blood of the recipient. Such formulationssuitably comprise a solution of a pharmaceutically and pharmacologicallyacceptable salt of oxidised thymosin β4, that is isotonic with the bloodof the recipient.

Useful formulations also comprise concentrated solutions or solidscontaining oxidised thymosin β4, which upon dilution with an appropriatesolvent give a solution for parental administration as above.

The oxidised thymosin β4 or physiologically active variant thereofdisclosed herein may be administered to the lungs of a subject by anysuitable means, but are preferably administered by generating an aerosolcomprised of respirable particles, the respirable particles comprised ofthe active compound, which particles the subject inhales (i.e., byinhalation administration). The respirable particles may be liquid orsolid.

Particles comprised of oxidised thymosin β4 for practising the presentinvention should include particles of respirable size: that is,particles of a size sufficiently small to pass through the mouth andlarynx upon inhalation and into the bronchi and alveoli of the lungs. Ingeneral, particles ranging from about 0.5 to 10 microns in size (moreparticularly, less than about 5 microns in size) are respirable.Particles of non-respirable size which are included in the aerosol tendto deposit in the throat and be swallowed, and the quantity ofnon-respirable particles in the aerosol is preferably minimized. Fornasal administration, a particle size in the range of 10-500 μm ispreferred to ensure retention in the nasal cavity.

Liquid pharmaceutical compositions or oxidised thymosin β4 for producingan aerosol can be prepared by combining the oxidised thymosin β4 with asuitable vehicle, such as sterile pyrogen free water. Solid particulatecompositions containing respirable dry particles of micronized oxidisedthymosin β4 may be prepared by grinding dry oxidised thymosin β4 with amortar and pestle, and then passing the micronized composition through a400 mesh screen to break up or separate out large agglomerates. A solidparticulate composition comprised of the oxidised thymosin β4 mayoptionally contain a dispersant which serves to facilitate the formationof an aerosol. A suitable dispersant is lactose, which may be blendedwith the oxidised thymosin β4 in any suitable ratio (e.g., a 1 to 1ratio by weight).

Aerosols of liquid particles comprising the oxidised thymosin β4 may beproduced by any suitable means, such as with a nebulizer. See, e.g.,U.S. Pat. No. 4,501,729. Nebulizers are commercially available deviceswhich transform solutions or suspensions of the oxidised thymosin β4into a therapeutic aerosol mist either by means of acceleration of acompressed gas, typically air or oxygen, through a narrow venturiorifice or by means of ultrasonic agitation. Suitable compositions foruse in nebulizers consist of the oxidised thymosin β4 in a liquidcarrier, the oxidised thymosin β4 comprising up to 40% w/w of thecompositions, but preferably less than 20% w/w. the carrier is typicallywater or a dilute aqueous alcoholic solution, preferably made isotonicwith body fluids by the addition of, for example, sodium chloride.Optional additives include preservatives if the composition is notprepared sterile, for example, methyl hydroxybenzoate, antioxidants,flavouring agents, volatile oils, buffering agents and surfactants.

Aerosols of solid particles comprising the oxidised thymosin β4 maylikewise be produced with an solid particulate medicament aerosolgenerator. Aerosol generators for administering solid particulatemedicaments to a subject produce particles which are respirable, asexplained above, and generate a volume of aerosol containing apredetermined metered dose of a medicament at a rate suitable for humanadministration. Examples of such aerosol generators include metered doseinhalers and insufflators.

For inflammation of external tissues, e.g. skin, the formulations arepreferably applied as a topical ointment or cream containing the activeingredient in an amount of, for example, 0.075 to 20% w/w, preferably0.2 to 15% w/w and most preferably 0.5 to 10% w/w. When formulated in anointment, the active ingredients may be employed with either aparaffinic or a water-miscible ointment base. Alternatively, the activeingredients may be formulated in a cream with an oil-in-water creambase.

If desired, the aqueous phase of the cream may include, for example, atleast 30% w/w of a polyhydric alcohol, i.e. an alcohol having two ormore hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glyercol and polyethylene glycol and mixturesthereof. The topical formulations may desirably include a compound whichenhances absorption or penetration of the active ingredient through theskin or other affected areas. Examples of such dermal penetrationenhancers include dimethylsulphoxide and related analogues.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner. While the phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Preferably, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and/or fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyle alcohol, glycerol mono-stearate and sodium lauryl sulphate.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties, since the solubility of theactive compound in most oils likely to be used in pharmaceuticalemulsion formulations is very low. Thus the cream should preferably be anon-greasy, non-staining and washable product with suitable consistencyto avoid leakage from tubes or other containers. Straight or branchedchain, mono-or dibasic alkyl esters such as di-isoadipate, isocetylstearate, propylene glycol diester of coconut fatty acids, isopropylmyristate, decyl oleate, isopropyl palmitate, butyl stearate,2-ethylhexyl palmitate or a blend of branched chain esters known asCrodamol CAP may be used, the last three being preferred esters. Thesemay be used alone or in combination depending on the propertiesrequired. Alternatively, high melting point lipids such as white softparaffin and/or liquid paraffin or other mineral oils can be used.

In addition to the aforementioned ingredients, the formulations of thisinvention may further include one or more accessory ingredient(s)selected from diluents, buffers, flavouring agents, binders, surfaceactive agents, thickeners, lubricants, preservatives (includingantioxidants) and the like.

The following examples describe the purification of the peptide factorand the characterisation of partially purified factor andsteroid-treated monocyte supernatant. Later examples describe thecharacterisation of purified factor as oxidised thymosin and activity ofthe oxidised thymosin β4.

BRIEF DESCRIPTION OF THE DRAWINGS

The examples are described with reference to the accompanying figures,wherein:

FIG. 1 illustrates the effects of STMS on human neutrophil locomotion.Cells were tracked for 2 minutes using the computerised tracking assaymethod. The mean value of cell speed determined at 5 second intervalswas 15.2 μm/min for speed, and extrapolated values of 1.02 μm²/sec and63 secs. for the diffusion coefficient and persistence respectively.

FIG. 2 illustrates the effect of supernatants from human monocytescultured for 24 hours at 37° C., with and without 10⁻⁶M dexamethasone,on the adhesion of human neutrophils to bovine aorta endothelial cellmonolayers. (a) culture medium without dexamethasone; (b) culture mediumwith 10⁻⁶M dexamethasone; (c) control monocyte supernatant (CMS); (d)steroid-treated monocytesupernatant (STMS). Mean±s.e.m. (verticalbars)n=3.P<0.001 given by **.

FIG. 3 illustrates the comparison of the morphology of human neutrophilstreated with STMS and various neutrophil locomotion stimulators, usingscanning electron microscopy. (a) 10⁻⁸M fMLP; (b) IL-8 and (c) STMS.Bar2 μm.

FIG. 4 illustrates the comparison of the morphology of human neutrophilstreated with STMS and various neutrophil locomotion stimulators andseeded on bovine aorta endothelial cell monolayers, using scanningelectron microscopy. (a) 10⁻⁸M fMLP and (b) STMS. Bar 2 μm.

FIG. 5 illustrates the comparison of F-actin distribution in humanneutrophils treated with STMS and various neutrophil locomotionstimulators. (a) culture medium with and without 10⁻⁶M dexamethasone;(b) 10⁻⁸M fMLP: (c)TNF; (d) STMS. Bar 2 μm.

FIG. 6 illustrates the inhibition of fMLP-induced chemotaxis of humanneutrophils by STMS measured using a modified Boyden chamber assay. (a)upper chamber—culture medium with dexamethasone; lower chamber—10⁻⁹fMLP; (b) upper chamber—STMS; lower chamber—10⁻⁹M fMLP; (c) upperchamber—10⁻⁹M fMLP; lower chamber—10⁻⁹M fMLP: (d) upper chamber—culturemedium with dexamethasone; lower chamber—STMS. Number of cells whichhave migrated half of the mean migration distance of positive control(a). □ Cell-front migration distance. Five randomly selected fields werecounted for each filter. Values shown are mean±s.e.m. (verticalbars)P<0.001 given by **.

FIG. 7 shows the data for peptide 1 showing the observed ion series forlow-energy CID following derivatisation with SPA. Key: Coff Collisionoffset (volts); Xle Leucine (Leu) or Isoleucine (Ile); Kpy Lysineepsilon-N-(3-pyridyl) acetate; Mso methionine sulphoxide; and *indicates ions observed in CID spectrum.

FIGS. 8a-d illustrate (a) dispersive locomotion of neutrophils inresponse to thymosin β4 (Tβ4) and oxidised thymosin β4 (Tβ4so); (b) FMLPinduced chemotaxis of human neutrophils in a modified Boyden chamber isinhibited by Tβ4so to a higher degree than the non-modified peptide. Adose dependent effect was observed of inhibition of chemotaxis by Tβ4and Tβ4so, with the oxidised peptide being tenfold more inhibitory thanthe native peptide; and (c) thymosin β4 sulphoxide promotes wouldhealing in a simple scratch assay. Oxidising the methionine residue (met6) was shown to increase the closure rate of scratch made on anendothelial monolayer on tissue culture plastic. (d) thymosin β4promotes would healing in a simple scratch assay. Oxidising themethionine residue (met 6) was shown to increase the closure rate ofscratch made on an endothelial monolayer on tissue culture plastic.

FIGS. 9a-9 c show the results of assays of Tβ4 and Tβ4so in theCarrageenan induced oedema test.

EXAMPLES

Materials and Methods

Reagents

IL-8 and TNF were purchased from Genzyme, dissolved in phosphatebuffered saline (PBS) and stored at 1 μg per ml at −70° C. Dexamethasoneand fMLP were purchased from Sigma Chemical Co. Dexamethasone wasprepared as a 10⁻²M stock solution in ethanol and fMLP as a 10⁻²M stocksolution in dimethyl sulphoxide (DMSO).

Neutrophil Purification

Neutrophils were obtained as described by Chettibi et al. [1993].Briefly, whole blood was mixed with 1:10 v/v of 5% dextran and allowedto sediment at 37° C. for 1 hour. The leucocyte rich plasma was layeredover Nycoprep 1.077 and centrifuged at 750 g for 15 minutes.Erythrocytes in the resulting pellet were removed by hypotonic lysis andneutrophils were washed twice with balanced salt solution (BSS). Thecells were checked for viability by trypan blue exclusion (generallygreater than 96% viable).

Cell Tracking Assay

Automated cell-tracking migration chambers were made as previouslydescribed [Chettibi et al., 1993] and placed on the stage of an invertedphase-contrast microscope within a temperature controlled (37° C.)transparent box and locomotion observed by means of a video cameraconnected to a monochrome monitor and also to an Acorn A5000 computerwith a Watford video digitiser programmed to capture and analyze oneframe every 5 seconds. Data was obtained from a maximum of 80 selectedcells and used to calculate instantaneous speed, the 2-dimensionaldiffusion coefficient and the locomotion persistence time, using thedescribed procedure to eliminate the contribution of systematic drift[Chettibi et al., 1994].

Adhesion Assay

Bovine aorta endothelial cells were cultured on 13 mm diameter glasscoverslips in a multi-well dish in Dulbeccols modified Eagles mediumwith 10% foetal calf and 10% horse serum and grown to confluence. Humanneutrophils suspended in BSS 0.1% bovine serum albumin (BSA) werelabelled with [⁵¹Cr]sodium chromate by incubating them at 1×10⁶ cells/mlfor 1 hour, 20 μCi/ml with periodic agitation. Free ⁵¹Cr was removed bythree washes with BSS 0.1% BSA. 200 μl of neutrophils were mixed with800 μl of STMS peptide factor or other test substances, added to thewells and incubated for 30 minutes at 37° C. Non-adherent cells werewashed gently three times with BSS 0.1% BSA and coverslips were placedin a Wilj gamma counter.

Electron Microscopy

Neutrophils were stimulated with various agonists for 20 minutes beforefixing in 2% buffered gluteraldehyde for one hour and washed twice inPBS. Post-fixation in osmium tetroxide was followed by washing indistilled water. Uranyl acetate was then added to the samples and leftin the dark for at least one hour before washing. The cells were passedthrough a graded series of acetone (or alcohols) ranging from 30% todried absolute, before critical point drying and mounting.

An alternative to critical point drying was lyophilization in which,after the osmium tetroxide had been washed from the cells, they wereplunged into liquid nitrogen (only suitable for cells adherent to asolid substrate). The specimens were then coated in gold and viewed inthe scanning electron microscopy (SEM).

Actin Staining and Confocal Microscopy

Purified neutrophils were placed on albumin-coated glass coverslipsbefore treatment with various stimuli and incubated at 37° C. for 30minutes. The cells were fixed in 1% paraformaldehyde solution for onehour, washed with BSS and permeabilised with 1% Triton x-100 for 15minutes at room temperature. Cells were washed three times with BSS andtreated with 0.1 mg/ml TRITC labelled phalloidin for 20 minutes at roomtemperature. Cells were washed three times in BSS at 5 minutes intervalsand mounted on glass slides with 50% glycerol. Results were analyzedusing confocal microscopy.

Chemotaxis Assay

Filters (Sartorius membrane filter 3 μm pore) were cut and glued to amodified iml syringe barrel. 300 μl of 2×10⁶/ml neutrophils were addedto 300 μl antagonist and 200 μl of the suspension was added to the upperchamber (syringe barrel). The lower chamber (a 5 ml beaker), contained3.6 ml of agonist. After 45 minutes, the cells were fixed in 70% ethanolfor 5 minutes. This procedure also removes the filter from the syringebarrel by dissolving the glue. The filters were placed in a multi-welldish and treated as follows: distilled water for 2 minutes, Harrishaematoxylin 1 minute, distilled water 1 min, Scotts Tap water (1:1 0.7%sodium bicarbonate:4% Magnesium sulphate (v/v) for 5 minutes, 70%ethanol 3 mins, 95% ethanol 3 minutes and 80%:20% ethanol:butanol (v/v)5 minutes. The filters were cleared in xylene for 5 minutes, mounted inDEPEX and examined under bright field illumination with a 40× objective.Five randomly selected fields were counted for each filter.

Scratch wound assay: Human umbilical vein endothelial cells (HUVEC) weregrown to confluency in multiwell dishes (Corning) and a scratch madeacross the diameter of each well with a sterile pipette tip. Theresulting wound (approx. 1 mm) was then measured before the addition ofTβ4 or Tβ4so and at 45 min intervals.

Induction of Inflammatory Response to Carrageenin: Groups of mice wereinjected subcutaneously in one hind paw with 300 μg carrageenin mixedthe thymosin β4, native or sulphoxide in a final volume of 50 μl.Control animals were injected with the same volume of saline. Footpadswelling was measured using a spring-dial calliper, and expressed as thedifference in swelling between the carrageenin-injected paw and theuninjected, contralateral paw. The animals were injectedintraperitoneally (i.p.) With the same dose of thymosin β4, native orsulphoxide and footpad measurements made at 6, 24, 48 and 72 hours.

Example 1

Preparation of Steroid Treated Monocyte Supernatant (STMS) and PartiallyPurified STMS Peptide Factor

Steroid treated monocyte supernatant (STMS) was obtained by the cultureof human monocytes which had been plated out in Hams F-10 medium at aconcentration of 5×10⁷ cells per ml in the presence of heat-inactivated10% foetal calf serum (FCS) for 60 mins, rinsed with Phosphate bufferedsaline (PBS) and then cultured in the absence of FCS for 24 hours in thepresence of 10⁻⁶M dexamethasone.

STMS Peptide Factor Preparation

STMS was obtained essentially as described by Chettibi et al. [1993] bythe culture of human monocytes in Hams F-10 medium with 10% foetal calfserum (FCS) at a concentration of approximately 5×10⁷ cells per ml for60 minutes, rinsed with PBS, and then cultured without FCS for 24 hoursin the presence of 10⁻⁶M dexamethasone. Parallel cultures in whichdexamethasone was omitted were used to prepare control monocytesupernatant (CMS). Purification of STMS peptide factor was carried outusing the 2-dimensional diffusion coefficient to identify activefraction. Partial purification was achieved using gel filtration andion-exchange chromatography on mono-Q resin. Highly purified materialwas obtained by the additional use of reverse phase HPLC.

Initial sequence analysis of the peptide factor was unsuccessful becauseof a presumed blocked N-terminal.

These observations suggested that treatment of neutrophils with STMSinduced a highly unusual mode of cytoskeletal organisation (FIG. 5d),but did not cast any direct light on the underlying basis for persistentlocomotion. The apparent correlation of behaviour with adhesion underthe light microscope was therefore extended by scanning EM and confocalmicroscopy studies.

Example 2

Biological Studies of STMS Peptide Factor

The biological interest in STMS Peptide Factor lies in its potentialrole as a mediator of some or all of the anti-inflammatory effects ofglucocorticoids. Many preliminary observations using the supernatant asopposed to the peptide factor seemed to support this role, but others,such as the phenomenon of dispersive locomotion, were not obviousanti-inflammatory responses. However, lowered adhesiveness, whichappears to be one of the underlying causes of dispersive locomotion, hasclear anti-inflammatory implications.

Characteristics of Neutrophil Locomotion

Agonists were used at concentrations which caused similar, sub-maximalstimulation of basal motility. Previous studies of crude and partiallypurified STMS peptide factor showed that it stimulated neutrophils toundergo highly dispersive locomotion in a uniform concentrationgradient. This was in marked contrast to responses to other agents andin particular to fMLP where the locomotor characteristics. suggestedthat the cells, though highly motile, could not readily break theirinitial adhesions to the substrate. Here it is shown that partiallypurified STMS also produces a dispersive response at a concentrationthat gives a similar instantaneous speed at 10 nm fMLP (FIGS. 1,2).

In addition to the determination of quantitative locomotor parameters,observation of the cells during the assay showed very clear andcharacteristic patterns of behaviour when the cells were treated withdifferent stimulating agents. Neutrophils exposed to STMS, rapidlybecome phase-dark corresponding to flattening and adhesion, but thenregain the phase bright state and become motile. In contrast IL-8induces characterised cyclic behaviour in which the cells darken andbrighten reversibly, whilst cells treated with fMLP remain phase bright.STMS treated cells also showed a very characteristic appearance givingthe subjective impression that the cell is attached to the substrate ata single site while the cell body is dynamically active above it. Allother stimulants tested appeared to cause the neutrophils to formseveral attachment points to the substrate. This observation isconsistent with previous adhesion studies which showed that STMS tracedneutrophils were very readily washed off a protein-coated glass surface.

Neutrophil Polarisation and Membrane Morphology

Neutrophils treated with partially purified STMS peptide factor appearedunder phase-contrast microscopy to be elongated and the characteristicsof their adhesion to a protein-coated glass surface indicated that theymight be attached at a single site. The cells were relatively easilydetached by washing and also the appearance was suggestive of largelyunattached cell body connected to the surface by an elongated process.

Scanning Electron Microscopy

The results of this study demonstrated that cells treated with fMLP orIL-8 showed classic polarisation with abundant areas of ruffled membraneand no clear leading edge (FIGS. 3a,b,c). In addition to the rufflingshown by the apparent points of contact with the substratum, themembrane over the body of the cell was highly convoluted. Differences inthe membrane induced by fMLP and IL-8, although apparent, were very hardto define. In contrast, the morphology induced by STMS was unique andreadily described (FIG. 3c).

The cells were of extended bipolar shape but the two ends that appearedto be involved in adhesive contact with the substratum were notidentical. The membrane was relatively smooth with numerous smallprotrusions and there was no very clear difference in surface appearancebetween the cell body and the apparent pseudopodia.

Neutrophils were seeded on bovine aorta endothelial monolayers for 30minutes and prepared for SEM. This treatment made little difference tothe appearance of the surface membrane which was ruffled in response toIL-8 and fMLP, but relatively smooth in response to STMS. The mostsignificant difference was that whereas fMLP and IL-8 treatedneutrophils were almost exclusively found at endothelial cell junctions,STMS treated cells tended to be found on the body of the endothelialcell (FIGS. 4a,b).

Confocal Microscopy

To investigate the underlying basis of the shape differences and tounderstand the nature of dispersive locomotion, we examined thedistribution of polymerised actin in the cells. Control non-activatedneutrophils showed a weak, rather punctate distribution of fluorescence,but with no sign of a major polarised focus corresponding to arelatively even distribution of cortical actin (FIG. 5a). The resultsobtained from fMLP were similar to those presented by other workers[Coates et al., 1992] and are consistent with the interpretation thatactin polymerisation is much more intense than in control cells and isassociated with points of adhesive contact that are foci of activelocomotion (FIG. 5b). Cells that were highly spread in response to CMSor to TNF showed an extremely punctate distribution of F-actin (FIG.5c). The pattern of actin staining in STMS-treated cells was highlyunusual and distinct. Staining was only present in the extremes of thebipolar cells and of these two ends, which appear to be points ofadhesive contact, one was invariably more intensely stained than theother (FIG. 5d).

Modulation of the Adhesion of Neutrophils to Endothelial Cells

Partially purified peptide factor reduced the adhesiveness ofneutrophils to an endothelial cell monolayer. Scanning EM studies ofneutrophil/endothelium interactions showed that in marked contrast toother stimuli, the supernatant peptide factor prevents adhesion andapparent invasion at endothelial cell junctions.

Inhibition of Neutrophil Secretion

Partially purified peptide factor inhibited the secretion of elastasefrom cytochalasin treated neutrophils (data not shown).

Example 3

Detailed Biological Studies of STMS and Peptide Factor NeutrophilAdhesion to Bovine Aorta Endothelial Cells

The previous data showing that STMS diminished neutrophil adhesion toprotein-coated glass [Chettibi et al., 1993] was extended using the morephysiological substrate of bovine aorta endothelial cells (FIG. 2).

Chemotaxis

Preliminary observations of neutrophil chemotaxis using the modifiedBoyden chamber revealed a striking contract between the response to STMSand fMLP. Neutrophils showed massive invasion of the filter when fMLPwas present in the lower chamber, but gave no such response to STMS.However when the filters were examined by the leading front method itbecame clear that a few STMS-treated cells were able to invadesuccessfully, in keeping with the predictions for persistent locomotion.These observations suggested that a more suitable analysis would be tomeasure total invasion, or to determine the number of cells presentmidway between the leading front and the surface of the filter. FIG. 6presents the analysis of chemotactic assays using both the leading frontand the average invasiveness methods. The results showed that STMS wasnot itself chemotactic, but when present in the upper chamber,dramatically inhibited the response to fMLP. In a uniform concentrationof fMLP (i.e. present in both chambers) was a marked reduction ininvasiveness.

Because the production of STMS is induced by anti-inflammatory steroids,there is reason to believe that this molecule may be a trueanti-inflammatory mediator. Most of the properties presented to date areconsistent with a role as inhibitor of the pro-inflammatory responses ofneutrophils, however its best characterised property, the induction ofpersistent locomotion in target cells, is by no means an anticipatedanti-inflammatory response. Pro-inflammatory cytokines have a complexprogramme of low-dose and high-dose effects which result in an extremelyvaried programme of responses for cells exposed to a concentrationgradient of such molecules. Many of the dynamic effects of fMLP forexample, are interpreted primarily in terms of response to aconcentration gradient. In contrast, the effect of STMS on neutrophilsdoes not indicate the quantitatively distinct effects would be observedat low and high concentration gradients. The studies with uniformagonist concentrations further emphasises the critical role ofconcentration gradients for the chemotactic response thus althoughneutrophils treated with a uniform concentration of fMLP or IL-8 havehighly active locomotory processes, they are unable to make effectivedisplacement. In contrast a similar degree of locomotory activitydisplayed by STMS treated cells leads to a highly effective locomotion.

It is well established that actin polymerisation is an early response tostimulation of neutrophils by TMLP. Coates et al. [1992] have providedevidence that actin polymerisation is the early and dominant event indetermining shape changes and dictating the specific patterns ofpolarisation. The link between the fMLP receptor and these earlymembrane events is believed to be provided by protein kinase C (PKC)activation that then leads to activation of the small cytosolicGTP-binding proteins [Ridley, 1994].

In addition the role of PKC in the phosphorylation of MARCKs and thedynamic role of phosphorylation and calcium binding. in determining thecyclic interaction of actin filaments with the plasma membrane has beenrecently reviewed by Janmey, [1995]. The present observations suggestthat actin based locomotory processes are in competition around the cellperiphery and only lead to productive displacement if an agonistgradient causes unequal activity of such processes at the leading edgeof the cell. An alternative model is that the role of the gradient isprimarily to weaken adhesions at the tail of the cell.

The pattern of responses to STMS is unusual in many important respects.The actin pool appears to be highly dynamic, but the distribution ofF-actin is mainly constrained to one end giving a unipolar distributionof actin in a bipolar cell. The membrane over most of the cell bodyremains relatively smooth and this lack of ruffling was observed bothwith cells in suspension and on the surface of cells stuck (albeitloosely) to the endothelium. Interestingly, the lack of ruffles maymanifest itself in the inability of these neutrophils to discern thegaps between endothelial cells, thereby inhibiting an inflammatoryresponse.

The data obtained in the tilt assay, where cell locomotion is highlypolarised by a gravitational field, show that the active cells alwaysmaintain one point of stable adhesion with the substrate. It thereforefollows that to maintain this polarity the bulk of the polymerised actinmust cycle repeatedly from one end of the cell to the other. Thispattern of activity does tend to suggest that the dipolarity of the cellis determined by a pre-existing structural feature, as yetuncharacterised.

One of the most important aspects of the behaviour of STMS peptidefactor as an anti-inflammatory mediator is its modulation of cellresponses to pro-inflammatory mediators. This has been tested in theabove work using motility stimulation as the basic parameter forcomparison of activities. Under conditions where STMS and fMLP or IL-8are equipotent as locomotion simulators, the morphological, adhesive andlocomotor responses to STMS tend to dominate and the cells remainphase-bright, bipolar and undergo dispersive locomotion.

Of even greater significance, STMS Peptide Factor, which is not itselfchemotactic, is able to suppress the chemotactic response to fMLP. It isof interest here to discuss the role of persistence in chemotaxis. Bythe very nature of the concept, any form of directed locomotion haspersistence. This is seen most clearly in the case of the tilt assay[Chettibi et al., 1994] where the response to STMS showed more than 90%directional movement and the persistence although infinite, became anessentially meaningless concept. For locomotion in a uniformconcentration of STMS, persistence reflects some kind of inertia in thelocomotor system. Bearing in mind the low Reynolds number conditions,this inertia must relate to the organisation of the structure on whichthe cytoskeleton acts or redistributes itself during locomotion.

The electron micrographs of STMS-treated cells on an endothelial layershow firstly that the cells do not seek the junctions betweenendothelial cells or do not have suitable exploratory leading lamellaeto penetrate such a gap if encountered.

It can be concluded that persistence of neutrophil locomotion caused bysteroid-induced factor results from an effect on actin distribution andpolymerisation. This type of locomotion appears to render neutrophilsresistant to chemotactic stimuli and impairs their ability to adhere toendothelial cells and to migrate through endothelial cell junctions. Assuch this factor appears to be an important mediator ofanti-inflammatory glucocorticoid action and pure peptide factor may beeffectively used in place of steroids for therapy of chronicinflammatory conditions.

Example 4

Identification of Active Factor

STMS (Steroid Treated Monocyte Supernatant) was defined as an activityin the culture medium from steroid treated human monocytes thatinfluenced the motility of human neutrophils, specifically to givedispersive or persistent locomotion characterised by a high2-dimensional diffusion coefficient.

The material described by Dr Chettibi et al. was highly purified by acombination of ion-exchange and gel filtration steps, but the criticalprocedure was reverse phase HPLC using an HPLC columnand an elutiongradient made from

A) 0.1% trifluoroacetic acid

B) 0.1% trifluoroacetic acid in 50% acetonitrile

The activity eluted at or around 34% B.

Separation methods involving extremes of pH or ionic strength profoundlychanged the dose-response curve.

The most active material prepared by this method is light pink inconcentrated solution and was characterised by an elution trace showingan absorption peak at 214 nm with a distinct shoulder. Furtherresolution was not possible and neither could the activity be attributedto the peak or the shoulder. Mass spectroscopy showed a single majorcomponent of mass 1331 Da and fragmentation analysis gave species of1186 Da and 991 Da. Full analysis (Dr Pappin ICRF) confirmed that themajor peak was acyanocobalmin, a derivative of vitamin B₁₂. It wassurmised that vitamin B₁₂ may in fact be a contaminant or serving tomask the actual peptide factor. Preparation of STMS and subsequentpurification of the peptide factor was therefore conducted omittingvitamin B₁₂. It was at this time that it was discovered that cellsresponded to material eluting from HPLC with a concentration optimumthat was at least 10⁴-fold lower than the concentration present inculture medium. These observations suggested that the active factor wasa complex of a small molecule with a carrier protein.

Preparation of STMS undertaken omitting vitamin B₁₂ from the mediumresulted in a high yield of activity. This was purified using forcedialysis (in vacuo) to concentrate 500 ml of culture medium to less than20 ml. The dialisand was washed with 2 changes of distilled water. Thesample was then purified by absorption and elution from Mono Q anionexchange resin and gel filtration on the Pharmacia peptide column run athigh salt concentration. The sample was then subjected to purificationby reverse phase HPLC as above. The most highly purified material elutedas a single peak and was non-pigmented.

Mass spectroscopic analysis now revealed a single major peak of averagemass 4980 Da (+/−2 Da). Mass measurement following esterification of asmall portion of the peptide material indicated the presence of 11acidic amino acid residues (Aspartic and Glutamic acid).

Peptide was then digested with 100 ng trypsin (Boehringer, modified) in6 μl 50 mM ammonium bicarbonate (pH 7.8) containing 15% V/V n-propanol0.5% hexyl-B-glucopyranoside (HBG) overnight at 25° C. The digestedpeptides were then reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA)in order to enhance b-ion abundance and facilitate sequence analysis bytandem mass spectrometry (Sherman et al., 1995). Dried peptide fractionswere treated with 7 μl 1% w/v N-succinimidyl-2(3-pyridyl)acetate in 0.5MHEPES (pH 7.8 with NaOH) containing 15% v/v acetonitrile for 20 min onice. The reaction was terminated by 1 μl heptafluorobutyric acid (HFBA)and the solution immediately injected onto a capillary reversephasecolumn (300 um×15 cm) packed with POROS R2/H material (PerseptiveBiosystems, MA) equilibrated with 2% v/v acetonitrile/0.05% v/v TFArunning at 3 μl/min. The adsorbed peptides were washed isocraticallywith 10% v/v acetonitrile/0.05% v/v TFA for 30 minutes at 3 μl/min toelute the excess reagent and HEPES buffer. The derivatised peptides werethen eluted with a single step gradient to 75% v/v acetonitrile/0.1% v/vformic acid and collected in a single 4 μl fraction. Five derivatisedpeptides were then fully sequenced by low-energy collision-induceddissociation (CID) using a Finnigan MAT TSQ7000 triple quadrupole MSfitted with a nanoelectrospray source (Hunt et al., 1986; Wilm and Mann,1996). CID was performed using 2-3 mTorr argon with collisional offsetvoltages between −13V and −33V. The product-ion spectra were collectedwith Q3 scanned at 500 amu/sec.

The 5 sequences obtained were:

1) Ac-SDKPDMAE[LI]EKFDK Ac-acetyl; Met oxidised to the Met-sulphoxide(+16 Da)

2) TETQEK

3) NP[LI]PSK

4) ET[LI]EQEK

5) QAGES Free-Acid at C-terminus

The data identifying peptide 1 and confirming that the methionine wasoxidised to the met-sulphoxide are shown in FIG. 7.

Note: Cannot distinguish between Leu and lle [LI] as they are isomers.Sequences corresponded exactly to tryptic fragments of human ThymosinBeta-4.

Thymosin β4 was now prepared from human neutrophils by the method of(Hannapell et al 1982) which used HPLC purification of the perchloricacid supernatant. Four major peaks were obtained and analysed by massspectroscopy. The identification of two of these peaks was confirmed asThymosin β4 (the major peak) eluting at 37% B and oxidised thymosin β4eluting at 34% B. Two other peaks gave no MW signatures.

Thymosin β4 was also synthesised by Dr Pappin but the final product hadan unidentified modification believed to lie in the C-terminal serine.

We now attempted to oxidise the thymosin β4 using hydrogen peroxide andwith the HPLC elution pattern as the assay. Treatment of thymosin β4with H O (50 vol.) for five minutes at room temperature gave virtuallyquantitative conversation.

Dr Pappin showed this molecule to have average mass 4980 Da (+/−2 Da).

Example 5

The activity of Tβ4so, in the neutrophil locomotion assays showed Tβ4soas being dispersive at low concentrations and stimulating non-dispersivelocomotion above a concentration optimum, FIG. 8a. Native Tβ4 did notgive significant dispersive locomotion in these assays. Neutrophillocomotion assays are notoriously hard to standardise and we thereforeattempted to confirm the identity of the factor using an independentassay, the inhibition of chemotaxis. Earlier work had shown that STMSinhibited neutrophil chemotaxis to fMLP (Young et al 1997). We nowmeasured the activities of Tβ4 and Tβ4so, in the Boyden Chamber assayand showed that neither was chemotactic, but both inhibited chemotaxisto fMLP, with the oxidised form being an order of magnitude more potent,FIG. 8b. These results provided grounds to believe that Tβ4so might bethe biologically active extracellular form of Tβ4 and we investigatedthis by comparing the activity of the two species in the endothelialsheet wound closure test, one of the more accessible bioassays for Tβ4activity (Malinda et al 1997). The results for the effect of Tβ4 were inclose agreement with published data, but Tβ4so was active with at leastan order of magnitude higher potency than the native peptide, FIG. 8c.In view of the contamination of the native Tβ4 with the more activeoxidation product (7% in this case), the present results are consistentwith the hypothesis that Tβ4so is the sole biologically active species.In further studies HPLC purified Tβ4 was dried and stored under nitrogenat −20° C. and dissolved immediately before use. These results, and inparticular the inhibition of chemotaxis, gave reason to believe, thatTβ4so could attenuate neutrophil associated inflammatory processes, sothe in vitro observations were extended in vivo using thecarrageenan-induced inflammation model, which is characterised bymassive neutrophil infiltration with accompanying oedema formation(Ianaro 1994). Administration of Tβ4so 30 minutes prior to, during and 6hours after hind footpad injection of carrageenan into BALB/c miceinduced significant suppression of swelling, which was evident after 6 hand sustained up to 24 hours (FIG. 9a). Suppression was dose responsiveand specific, since administration of 10 or 100 ng doses of Tβ4so, or ofPBS of Tβ4 (1000 ng) was ineffective (FIG. 9b) A comparison between 800ng doses of Tβ4so and Tβ4 was carried out in this assay and clearlyshowed that only the oxidised peptide significantly reducedcarrageenan-induced oedema (FIG. 9c). These data clearly indicated thatmethionine oxidation was critical to achieve in vivo anti-inflammatoryactivity of Tβ4so, and strongly supported the biological plausibility ofthe in vitro findings.

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What is claimed is:
 1. A method of treating an inflammatory condition ina subject comprising administering an oxidised thymosin β4,physiologically active variant, or salt thereof.
 2. The method accordingto claim 1 wherein the inflammatory condition is due to a inflammatoryarthropathy, connective tissue disease, vasculitic syndrome, respiratorydisease, dermatological disease, gastrointestinal disease,haematological disease, transplantation/prosthetic rejection orinfection.
 3. The method according to claim 1 wherein the inflammatorycondition is a result of a separate drug therapy.
 4. A method oftreating sepsis in a subject comprising administering a therapeuticamount of an oxidised thymosin β4, physiologically active variant, orsalt therof.